Summary of Work: The stability of genomes, particularly human, is influenced by opportunities for mutations involving various unstable repeats. These repeats are abundant in the genome and represent at-risk motifs (ARMs) because of the high potential for genetic change. ARMs have also enabled us to detect subtle defects in DNA metabolic genes, which is important in considering human polymorphic variants that might have a small effect or that might exhibit strong synergistic interactions between alleles or with environmental factors. To characterize the roles of genes affecting genome stability and possible interactions between mutants,we examined the consequences of site- or regionally-directed mutations in domains and/or motifs identified in biochemical and structural studies. Several DNA metabolic defects were identified that exhibited strong pairwise synergistic interactions.i) A novel POL2 (DNA Pol epsilon) mutator allele was found that shows strong mutator effects when combined with an exo1-null, msh2 and pol-delta mutations. We demonstrated that the functional deficiency caused by this mutation is different from a defect in proofreading exonuclease of pol epsilon. Moreover, this mutation which results in +1 frameshifts in long homonucleotide runs corresponds to a new class of functional defects. ii) We found that a mutation in a RAD27/FEN1 gene that disrupts its interaction with PCNA appears to have little effect on genome stability; however, it exhibited synergy with defects in double- strand break repair. We discovered an extremely strong negative interaction (cell death) between this neutral allele of RAD27/FEN1 and a subtle mutator defect resulting from a heterozygous deficiency in the DNA polymerase delta 3 to 5 exonuclease. Our most recent experiments (as part of IRA effort with the Kunkel lab) have determined that mutations in the exonuclease domain, but not other domains, lead to this interaction. These results demonstrate a novel role for the exonucleolytic activity of DNA polymerase delta in addition to its function in proofreading errors that arise during replication. - DNA, DNA Recombinant, Gene Conversion, Genetic Markers, Radiation, Ionizing, Mitosis